Hyperactive piggyBac transposase integration efficiency
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Solution Overview
Problem
Current transposases and transposons, such as piggyBac, do not achieve sufficient integration efficiency for the generation of recombinant CHO cell lines, requiring extensive screening and time for high producer cell line development.
Innovation Solution
Development of a hyperactive piggyBac transposase with specific amino acid substitutions and modified left internal repeat sequences within transposable elements to enhance integration efficiency and reduce viability recovery time during selection.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If wild-type piggyBac transposase is used, then the transposon can integrate into the genome, but the integration efficiency is insufficient requiring extensive screening
Solution Approach 1:
The patent applies parameter changes by introducing specific amino acid substitutions at positions 30, 118, 185, 282, and 538 in the piggyBac transposase sequence. These substitutions modify the enzymatic parameters to enhance切割 (cutting) and integration activity, directly improving integration efficiency without requiring extensive screening of cell lines.
Solution Approach 2:
The patent creates a composite transposase system by combining multiple amino acid substitutions (I30A, Q118P, M185V, M282L, N538R) within the transposase protein structure. This composite modification approach synergistically enhances the overall transposase activity and integration efficiency beyond what single mutations achieve.
2Productivity
If standard transposon integration is used, then cell lines can be generated, but the process is time- and labor-intensive
Solution Approach 1:
The patent modifies the transposable element structure by introducing nucleotide substitutions in the left internal repeat sequence (positions 3, 9, 10, 12) to increase homology with the left repeat sequence. This structural parameter change enhances transposase recognition and integration efficiency, simplifying the overall cell line generation process.
3Reliability
If wild-type transposase is used, then integration occurs at TTAA sites, but the fraction of integrated cells is low
Solution Approach 1:
The patent introduces a combination of amino acid substitutions (I30A, Q118P, M185V, M282L, N538R) that collectively enhance the transposase's catalytic efficiency and stability. These parameter changes increase the fraction of cells achieving integration while maintaining reliable TTAA site targeting, resolving the contradiction between reliability and productivity.
Data Source
AI summary
The present invention relates to a polypeptide comprising a piggyBac transposase or a fragment or a derivative thereof having transposase function comprising at least one amino acid substitution. Further, the present invention relates to a transposable element comprising a piggyBac or piggyBac-like left repeat sequence and left internal repeat sequence, wherein the left internal repeat sequence comprises at least one nucleotide modification. Furthermore, the present invention relates to a kit comprising the above transposase and/or transposable element. In addition, the present invention relates to a targeting system comprising the above transposase and/or transposable element.